Protein production method

ABSTRACT

This invention relates to a method for producing a protein of interest, comprising introducing a protein expression vector which comprises a gene fragment a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell; integrating the gene fragment inserted between a pair of the transposon sequences, into a chromosome of the mammalian cell to obtain a mammalian cell capable of expressing the protein of interest; and suspension-culturing the mammalian cell; and a suspension mammalian cell capable of expressing the protein of interest.

This application is a Continuation of U.S. application Ser. No.14/689,782, filed Apr. 17, 2015 (now allowed); which is a Continuationof U.S. application Ser. No. 12/813,920 filed Jun. 11, 2010 (now U.S.Pat. No. 9,034,649); which claims the benefit of U.S. ProvisionalApplication No. 61/186,138, filed Jun. 11, 2009; and which claimspriority to Japanese Patent Application No. 2009-140626, filed Jun. 11,2009; the contents of all of which are incorporated herein by referencein their entireties.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to a method for producing a protein of interest,comprising introducing a protein expression vector which comprises agene fragment comprising a DNA encoding a protein of interest and aselectable marker gene and transposon sequences at both terminals of thegene fragment, into a suspension mammalian cell, integrating the genefragment inserted between a pair of the transposon sequences into achromosome of the mammalian cell to obtain a mammalian cell capable ofexpressing the protein of interest; and suspension-culturing themammalian cell; and a suspension mammalian cell capable of expressingthe protein of interest.

2. Brief Description of the Background Art

Production of exogeneous proteins by recombinant DNA techniques is usedin various industries such as pharmaceutical industry and food industry.In most cases, production of recombinant proteins is carried out byintroducing an expression vector comprising a nucleotide sequenceencoding a protein of interest into a host, such as Escherichia coli,yeast, insect cell, plant cell, and animal cell, selecting atransformant in which the expression vector is integrated into thechromosome, and further culturing the cell line under appropriateculture conditions.

However, in order to develop a host which can produce an exogeneousprotein efficiently, it is necessary to select a host cell having goodproductivity for each protein of interest, so that a further technicalinnovation is desired on the exogeneous protein production techniquesfor individual host.

In the bacteria systems, such as Escherichia coli, and yeast systems,different from animal cells, post-translational modifications, such assugar chain modification, are difficult to attain in many cases and thuscause a problem in producing a protein having its activity.

Since the produced protein is subject to a post-translationalmodification such as phosphrylation and addition of sugar chains in theinsect system, this system has a merit that the protein having itsoriginal physiological activity can be expressed. However, since thesugar chain structure of the secreted protein is different from that ofmammalians-derived cells, antigenicity and the like become a problemwhen the protein is applied to pharmaceutical use.

In addition, since a recombinant virus is used in the insect cell systemwhen an exogeneous gene is introduced, there is a problem that itsinactivation and containment of the virus are required from theviewpoint of safety.

In the animal cell system, post-translational modifications, such asphosphorylation, sugar chain addition, and folding, can be conducted toproteins derived from higher animals including human, in more similarmanner to those produced in the living body. Such accuratepost-translational modifications are necessary for recreating thephysiological activity originally possessed by a protein in itsrecombinant protein, and a protein production system in which amammalian cell is used as a host is usually applied to pharmaceuticalproducts and the like that requires such physiological activity.

However, a protein expression system in which a mammalian cell is usedas the host is generally low in productivity, and also causes a problemof the stability of introduced genes in many cases. Improvement ofproductivity of a protein using a mammalian culture cell as a host isnot only very important in producing medicaments for treatment,diagnostic agents and the like, but also greatly contributes to researchand development of them. Thus, it is urgent to develop a gene expressionsystem which easily makes it possible to obtain a cell line of a highproductivity using a mammalian culture cell, particularly Chinesehamster ovary cell (CHO cell), as the host.

A transposon is a transposable genetic element which can transfer fromone locus to other locus on the chromosome. A transposon is a strongtool for the study on molecular biology and genetics and used for apurpose, such as mutagenesis, gene trapping, and preparation oftransgenic individuals, in insects or nematode (e.g., Drosophilamelanogaster or Caenorhabditis elegans) and plants. However, developmentof such a technique has been delayed for vertebral animals includingmammalian cells.

In recent years, however, transposons which have activities also invertebral animals have been reported, and some of them were shown tohave an activity in mammalian cells, such as cell derived from mouse andhuman. Typical examples include transposons Tol1 (Patent Reference 1)and Tol2 (Non-patent Reference 1) cloned from a medaka (killifish),Sleeping Beauty reconstructed from a non-autonomous transposon existedin Onchorhynchus fish genome (Non-patent Reference 2), an artificialtransposon Frog prince (Non-patent Reference 3) which is derived fromfrog and a transposon piggyBac (Non-patent Reference 4) which is derivedfrom insect.

These DNA transposons have been used for mutagenesis, gene trapping,preparation of transgenic individuals, expression of drug-resistantproteins, and the like, as a gene transfer tool for bringing a newphenotype in a genome of a mammalian cell (Non-patent References 5 to12).

In the case of insects, a method in which an exogeneous gene isintroduced into silkworm chromosome using the transposon piggyBacderived from a Lepidoptera insect to express the protein encoded by saidexogeneous gene was studied, and a protein production method using theabove techniques has been disclosed (Patent Reference 2).

However, since the expressed protein of interest is not sufficient inexpression level and is produced in the whole body of silkworm, itcauses an economical problem due to the necessity of an advancedpurification technique for recovering the expressed exogeneous proteinin a highly purified form from the body fluid including a large amountof contaminated proteins.

In addition, an example in which a protein relating to G418 resistanceis expressed in a mammalian cell using the medaka-derived transposonTol2 (Non-patent Reference 12) is known.

CITATION LIST Patent Literature

-   [Patent Literature 1] WO2008/072540-   [Patent Literature 2] Japanese Published Unexamined Patent    Application No. 2001-532188

Non Patent Literature

-   [Non Patent Literature 1] Nature 383, 30 (1996)-   [Non Patent Literature 2] Cell 91, 501-510 (1997)-   [Non Patent Literature 3] Nucleic Acids Res, 31, 6873-6881 (2003)-   [Non Patent Literature 4] Insect Mol. Biol. 5, 141-151 (1996)-   [Non Patent Literature 5] Genetics. 166, 895-899 (2004)-   [Non Patent Literature 6] PLoS Genet, 2, e169 (2006)-   [Non Patent Literature 7] Proc. Natl. Acad. Sci. USA 95, 10769-10773    (1998)-   [Non Patent Literature 8] Proc. Natl. Acad. Sci. USA 98:6759-6764    (2001)-   [Non Patent Literature 9] Nature 436, 221-226 (2005)-   [Non Patent Literature 10] Nucleic Acids Res., 31, 6873-6881 (2003)-   [Non Patent Literature 11] Nucleic Acids Res., 35, e87 (2007)-   [Non Patent Literature 12] Proc Natl. Acad. Sci. USA, 103,    15008-15013 (2006)

SUMMARY OF THE INVENTION

In order to produce and analyze a protein of interest, it is necessaryto select a cell line which stably and highly expresses a protein ofinterest, using a mammalian-derived culture cell, but preparation andculture of the cell that produces the protein of interest requireconsiderable labor and time.

In addition, though it is known that a protein of interest is expressedin a mammalian cell using a transposon sequence, preparation of a cellwhich can highly express a protein of interest and thus can be used as aprotein production system by using a transposon sequence; preparationmethod of a mammalian cell which can highly produce a protein ofinterest by using a transposon sequence; and a production method of aprotein using the cell are not known.

As described in the above, the expression of a protein of interest in alarge amount by establishing a protein production system which canhighly produce a protein of interest using a mammalian culture cellefficiently and within a short period has been required. Thus, theobjects of the invention are to provide a cell capable of highlyexpressing a protein of interest which can be efficiently established,and a method for producing the protein of interest using the cell.

Solution to Problems

To solve the above-mentioned problems, the present inventors haveconducted intensive studies and found as a result that a mammalian cellcapable of highly expressing a protein of interest can be efficientlyprepared by introducing a protein expression vector which comprises agene fragment comprising a DNA encoding the protein of interest and aselectable marker gene and transposon sequences at both terminals of thegene fragment, into a suspension mammalian cell; and integrating thegene fragment inserted between a pair (two) of the transposon sequencesinto a chromosome of the mammalian cell. In addition, it was found thatthe protein of interest can be produced efficiently by using the cell,and thereby the invention was accomplished.

According to the protein production method of the invention, a proteinof interest can be efficiently produced by the use of a mammalian cell.In addition, the cell of the invention can be used as a proteinproduction cell for producing a recombinant protein with a highefficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of a transposon vector forexpressing an anti-human influenza M2 antibody. Tol2-L represents a leftend Tol2 transposon (SEQ ID NO:2), Tol2-R represents a right end Tol2transposon (SEQ ID NO:3), CMV represents a CMV promoter, poly Arepresents a polyadenylation site, Hc represents a human antibody Hchain cDNA, Lc represents a human antibody L chain cDNA, and CHX-rrepresents a cycloheximide resistance gene.

FIG. 2 shows a schematic illustration of an anti-human influenza M2antibody expression vector. CMV represents a CMV promoter, poly Arepresents a polyadenylation site, Hc represents a human antibody Hchain cDNA, Lc represents a human antibody L chain cDNA and CHX-rrepresents a cycloheximide resistance gene.

FIG. 3 shows a schematic illustration of a Tol2 transposase expressionvector. CAGGS represents a CAGGS promoter, poly A represents apolyadenylation site, and TPase cDNA represents a Tol2 transposase cDNA.

FIG. 4A shows a result of examining expression level of an anti-humaninfluenza M2 antibody in a suspension CHO-K1 cell when a Tol2 transposonvector for expressing an anti-human influenza M2 antibody was used. Theordinate shows the amount of antibody production (μg/ml), and theabscissa shows the number of transgenic clones of the suspension CHO-K1cell.

FIG. 4B shows a result of examining expression level of an anti-humaninfluenza M2 antibody in an adhesive CHO-K1 cell when a Tol2 transposonvector for expressing an anti-human influenza M2 antibody was used. Theordinate shows the amount of antibody production (μg/ml), and theabscissa shows the number of transgenic clones of the adhesive CHO-K1cell.

FIG. 5 shows a schematic illustration of a Tol1 transposon vector forexpressing an anti-human influenza M2 antibody. Tol1-L represents a leftend Tol1 transposon (SEQ ID NO:14), Tol1-R represents a right end Tol1transposon (SEQ ID NO:15), CMV represents a CMV promoter, poly Arepresents a polyadenylation site, Hc represents a human antibody Hchain cDNA, Lc represents a human antibody L chain cDNA, and CHX-rrepresents a cycloheximide resistance gene.

FIG. 6 shows a schematic illustration of a Tol1 transposase expressionvector. CAGGS represents a CAGGS promoter, poly A represents apolyadenylation site, and TPase cDNA represents a Tol1 transposase cDNA.

FIG. 7 shows a result of examining expression level of an anti-humaninfluenza M2 antibody in a suspension CHO-K1 cell when a Tol1 transposonvector for expressing an anti-human influenza M2 antibody was used. Theordinate shows the amount of antibody production (μg/ml), and theabscissa shows the number of transgenic clones of the suspension CHO-K1cell.

DETAILED DESCRIPTION OF THE INVENTION

Specifically, the invention relates to the following 1 to 31:

1. A method for producing a protein of interest, comprising introducinga protein expression vector which comprises a gene fragment comprising aDNA encoding a protein of interest and a selectable marker gene andtransposon sequences at both terminals of the gene fragment, into asuspension mammalian cell; integrating the gene fragment insertedbetween a pair of the transposon sequences into a chromosome of themammalian cell to obtain a mammalian cell capable of expressing theprotein of interest; and suspension-culturing the mammalian cell;

2. A method for producing a protein of interest, comprising thefollowing steps (A) to (C):

(A) a step of simultaneously introducing the following expressionvectors (a) and (b) into a suspension mammalian cell:

(a) an expression vector which comprises a gene fragment comprising aDNA encoding a protein of interest and transposon sequences at bothterminals of the gene fragment,

(b) an expression vector which comprises a DNA encoding a transposasewhich recognizes the transposon sequences and has activity oftransferring a gene fragment inserted between a pair of the transposonsequences into a chromosome,

(B) a step of expressing transiently the transposase from the expressionvector introduced in the step (A) to integrate the gene fragmentinserted between a pair of the transposon sequences into a chromosome ofthe mammalian cell to obtain a suspension mammalian cell capable ofexpressing the protein of interest, and(C) a step of suspension-culturing the suspension mammalian cell capableof expressing the protein of interest obtained in the step (B) toproduce the protein of interest;

3. A method for obtaining a suspension mammalian cell capable ofexpressing a protein of interest, comprising introducing a proteinexpression vector which comprises a gene fragment comprising a DNAencoding a protein of interest and a selectable marker gene andtransposon sequences at both terminals of the gene fragment into asuspension mammalian cell; and integrating the gene fragment insertedbetween a pair of the transposon sequences, into a chromosome of themammalian cell;

4. The method described in any one of the aforementioned items 1 to 3,wherein the suspension mammalian cell is a cell capable of surviving andproliferating in a serum-free medium;

5. The method described in any one of the aforementioned items 1 to 4,wherein the suspension mammalian cell is at least one selected from asuspension CHO cell in which a CHO cell is adapted to suspensionculture, a PER.C6 cell, a rat myeloma cell YB2/3HL.P2.G11.16Ag.20 (oralso called YB2/0) and a suspension mouse myeloma cell NS0 adapted tosuspension culture;

6. The method described in the aforementioned item 5, wherein the CHOcell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO/DG44,Pro-3 and CHO-S;

7. The method described in any one of the aforementioned items 1 to 6,wherein the selectable marker gene is a cycloheximide resistance gene;

8. The method described in the aforementioned item 7, wherein thecycloheximide resistance gene is a gene encoding a mutant of humanribosomal protein L36a;

9. The method described in the aforementioned item 8, wherein the mutantis a mutant in which proline at position 54 of the human ribosomalprotein L36a is substituted with other amino acid;

10. The method described in the aforementioned item 9, wherein the otheramino acid is glutamine;

11. The method described in any one of the aforementioned items 1 to 10,wherein a pair of the transposon sequences are nucleotide sequencesderived from a pair of DNA-type transposons which function in amammalian cell;

12. The method described in the aforementioned item 11, wherein thenucleotide sequences derived from a pair of DNA type transposons arenucleotide sequences derived from a pair of Tol1 transposons ornucleotide sequences derived from a pair of Tol2 transposons;

13. The method described in the aforementioned item 12, wherein thenucleotide sequences derived from a pair of Tol2 transposons are anucleotide sequence comprising the nucleotide sequence shown in SEQ IDNO:2 and the nucleotide sequence shown in SEQ ID NO:3;

14. The method described in the aforementioned item 12, wherein thenucleotide sequences derived from a pair of Tol1 transposons are thenucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequenceshown in SEQ ID NO:15;

15. A suspension mammalian cell capable of producing a protein ofinterest, into which a protein expression vector comprising a genefragment comprising a DNA encoding a protein of interest and aselectable marker gene and transposon sequences at both terminals of thegene fragment is introduced, to integrate the gene fragment insertedbetween a pair of the transposon sequences into a chromosome;

16. A suspension mammalian cell capable of producing a protein ofinterest, into which an expression vector (a) comprising a gene fragmentcomprising a DNA encoding a protein of interest and a selectable markergene and transposon sequences at both terminals of the gene fragment,and an expression vector (b) comprising a DNA encoding a transposase (atransferase) which recognizes the transposon sequences and has activityof transferring the gene fragment inserted between a pair of thetransposon sequences into a chromosome to integrate the gene fragmentinserted between a pair of the transposon sequences into the chromosome;

17. The cell described in the aforementioned item 15 or 16, wherein thecell is a cell capable of surviving and proliferating in a serum-freemedium;

18. The cell described in any one of the aforementioned items 15 to 17,wherein the cell is at least one suspension mammalian cell selected froma suspension CHO cell in which a CHO cell is adapted to suspensionculture, a PER.C6 cell, a rat myeloma cell YB2/3HL.P2.G11.16Ag.20 (oralso called YB2/0) and a suspension mouse myeloma cell NS0 adapted tosuspension culture;

19. The cell described in the aforementioned item 18, wherein the CHOcell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO/DG44,Pro-3 and CHO-S;

20. The cell described in any one of the aforementioned items 15 to 19,wherein the selectable marker gene is a cycloheximide resistance gene;

21. The cell described in the aforementioned item 20, wherein thecycloheximide resistance gene is a gene encoding a mutant of humanribosomal protein L36a;

22. The cell described in the aforementioned item 21, wherein the mutantis a mutant in which proline at position 54 of the human ribosomalprotein L36a is substituted with other amino acid;

23. The cell described in the aforementioned item 22, wherein the otheramino acid is glutamine;

24. The cell described in any one of the aforementioned items 15 to 23,wherein a pair of the transposon sequences are nucleotide sequencesderived from a pair of DNA-type transposons which function in amammalian cell;

25. The cell described in the aforementioned item 24, wherein thenucleotide sequences derived from a pair of the DNA-type transposons arenucleotide sequences derived from a pair of Tol1 transposons ornucleotide sequences derived from a pair of Tol2 transposons;

26. The cell described in the aforementioned item 25, wherein thenucleotide sequences derived from a pair of the Tol2 transposons are thenucleotide sequence shown in SEQ ID NO:2 and the nucleotide sequenceshown in SEQ ID NO:3;

27. The cell described in the aforementioned item 25, wherein thenucleotide sequences derived from a pair of the Tol1 transposons are thenucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequenceshown in SEQ ID NO:15;

28. A protein expression vector, comprising a gene fragment comprising aDNA encoding a protein of interest and a selectable marker gene, and apair of transposon sequences at both terminals of the gene fragment;

29. The protein expression vector described in the aforementioned item28, wherein a pair of the transposon sequences are nucleotide sequencesderived from a pair of Tol1 transposons or nucleotide sequences derivedfrom a pair of Tol2 transposons.

30. The protein expression vector described in the aforementioned item29, wherein the nucleotide sequences derived from a pair of the Tol2transposons are the nucleotide sequence shown in SEQ ID NO:2 and thenucleotide sequence shown in SEQ ID NO:3; and

31. The protein expression vector described in the aforementioned item29, wherein the nucleotide sequences derived from a pair of the Tol1transposons are the nucleotide sequence shown in SEQ ID NO:14 and thenucleotide sequence shown in SEQ ID NO:15.

This invention relates to a method for producing a protein of interest,comprising introducing a protein expression vector comprising a genefragment comprising a DNA encoding a protein of interest and aselectable marker gene and transposon sequences at both terminals of thegene fragment, into a suspension mammalian cell; integrating the genefragment inserted between a pair (two) of the transposon sequences, intoa chromosome of the mammalian cell to obtain a mammalian cell capable ofexpressing said protein of interest; and suspension-culturing themammalian cell.

Examples of the method for producing a protein of interest of thepresent invention include a method, comprising the following steps (A)to (C):

(A) a step of simultaneously introducing the following expressionvectors (a) and (b) into a suspension mammalian cell:

(a) an expression vector which comprises a gene fragment comprising aDNA encoding a protein of interest and transposon sequences at bothterminals of the gene fragment,

(b) an expression vector which comprises a DNA encoding a transposasewhich recognizes the transposon sequences and has activity oftransferring a gene fragment inserted between a pair of the transposonsequences into a chromosome,

(B) a step of expressing transiently the transposase transiently fromthe expression vector introduced in the step (A) to integrate the genefragment inserted between a pair of the transposon sequences into achromosome of the mammalian cell to obtain a suspension mammalian cellcapable of expressing the protein of interest, and(C) a step of suspension-culturing the suspension mammalian cell capableof expressing the protein of interest obtained in the step (B) toproduce the protein of interest.

In addition, the present invention relates to a suspension mammaliancell capable of producing a protein of interest, into which a proteinexpression vector comprising a gene fragment comprising a DNA encoding aprotein of interest and a selectable marker gene and transposonsequences at both terminals of the gene fragment is introduced, tointegrate the gene fragment inserted between a pair of the transposonsequences into a chromosome.

Furthermore, the present invention relates to a suspension mammaliancell capable of producing a protein of interest, into which anexpression vector (a) comprising a gene fragment comprising a DNAencoding a protein of interest and a selectable marker gene andtransposon sequences at both terminals of the gene fragment, and anexpression vector (b) comprising a DNA encoding a transposase (atransferase) which recognizes the transposon sequences and has activityof transferring the gene fragment inserted between a pair of thetransposon sequences into a chromosome to integrate the gene fragmentinserted between a pair of the transposon sequences into the chromosome.

The term “transposon” in the present specification is a transposablegenetic element and means a gene unit which moves on a chromosome orfrom a chromosome to other chromosome (transposition) while keeping acertain structure.

The transposon comprises a gene unit of a repeating transposon sequences(also called inverted repeat sequence (IR sequence) or terminal invertedrepeat sequence (TIR sequence)) which positions in the same direction orthe reverse direction at both terminals of the gene unit and anucleotide sequence encoding a transposase which recognizes thetransposon sequence to transfer a gene existing between the transposonsequences.

The transposase translated from the transposon can transfer a DNA byrecognizing transposon sequences of both terminals of the transposon,cutting out the DNA fragment inserted between a pair of the transposonsequences and inserting the fragment into the site to be transferred.

The term “transposon sequence” in the present specification means thenucleotide sequence of a transposon recognized by a transposase and hasthe same meaning as the IR sequence or TIR sequence. A DNA comprisingthe nucleotide sequence may comprise an imperfect repeating moiety aslong as it can be transferred (inserted into other position in thegenome) by the activity of a transposase, and comprise a transposonsequence specific to the transposase.

As the transposon sequence to be used in the invention, a nucleotidesequence derived from a DNA-type transposon is preferable, and anucleotide sequence derived from a pair of natural or artificialDNA-type transposons, which can be recognized by a transposase and betransposed in mammalian cells, is more preferable.

Examples of the nucleotide sequence derived from a DNA-type transposoninclude the nucleotide sequences derived from the medaka fish-derivedTol1 transposon and Tol2 transposon, the Sleeping Beauty reconstructedfrom a non-autonomous transposon existed in an Onchorhynchus fishgenome, the frog-derived artificial transposon Frog prince and theinsect-derived transposon PiggyBac.

Particularly, among them, the nucleotide sequences derived from themedaka fish-derived Tol2 transposon comprising the nucleotide sequenceshown in SEQ ID NO:6 and the medaka fish-derived Tol2 transposoncomprising the nucleotide sequence shown in SEQ ID NO:13 are preferable.

Examples of the nucleotide sequence derived from a pair of Tol2transposons include the nucleotide sequence at positions 1 to 2229 andthe nucleotide sequence at positions 4148 to 4682 in the Tol2 transposonnucleotide sequence shown in SEQ ID NO:6 of Sequence Listing.

As the nucleotide sequence derived from a pair of Tol2 transposons, thenucleotide sequence at positions 1 to 200 (SEQ ID NO:2) (hereinafterreferred to as “Tol2-L sequence”) and the nucleotide sequence atpositions 2285 to 2788 (SEQ ID NO:3) (hereinafter referred to as “Tol2-Rsequence”) in the Tol2 transposon nucleotide sequence shown in SEQ IDNO:1 of Sequence Listing are more preferable.

Examples of the nucleotide sequence derived from a pair of Tol1transposons include the nucleotide sequence comprising a nucleotidesequence at positions 1 to 157 and the nucleotide sequence at positionsthe 1748 to 1855 in the Tol1 transposon nucleotide sequence shown in SEQID NO:13 of Sequence Listing.

As the nucleotide sequence derived from a pair of Tol1 transposons, thenucleotide sequence at positions 1 to 200 (SEQ ID NO:14) (hereinafterreferred to as “Tol1-L sequence”) and the nucleotide sequence atpositions 1351 to 1855 (SEQ ID NO:15) (hereinafter referred to as“Tol1-R sequence”) in the Tol2 transposon nucleotide sequence shown inSEQ ID NO:1 of Sequence Listing are more preferable.

Examples of the transposon sequence to be used in the invention includetransposon sequences of which transfer reactions are controlled by usinga partial sequence of a transposon sequence derived from theabove-mentioned transposon, by adjusting the length of the nucleotidesequence and by modifying the nucleotide sequence due to addition,deletion or substitution.

Regarding the control of the transfer reaction of a transposon, thetransfer reaction can be accelerated or suppressed by accelerating orsuppressing recognition of the transposon sequence by a transposase,respectively.

The term “transposase” in the present specification means an enzymewhich recognizes nucleotide sequences having transposon sequences andtransfers a DNA existing between the nucleotide sequences into achromosome or from the chromosome to other chromosome.

Examples of the transposase include the Tol1 and Tol2 which are derivedfrom medaka fish, the Sleeping Beauty reconstructed from anon-autonomous transposon existed in an Onchorhynchus fish genome, theartificial transposon Frog prince which is derived from frog and thetransposon PiggyBac which is derived from insect.

As the transposase, a native enzyme may be used, and any transposase inwhich a part of its amino acids are substituted, deleted, insertedand/or added may be used as long as the same transfer activity as thetransposase is maintained. By controlling the enzyme activity of thetransposase, the transfer reaction of the DNA existing between thetransposon sequences can be controlled.

In order to analyze whether or not it possesses a transfer activitysimilar to that of transposase, it can be measured by the 2-componentsanalyzing system disclosed in Japanese Published Unexamined PatentApplication No. 235575/2003.

Illustratively, whether or not a non-automatic Tol2 element can betransferred and inserted into a mammalian cell chromosome by theactivity of a transposase can be analyzed by separately using a plasmidcomprising a Tol2 transposase-deleted Tol2 transposon (Tol2-derivednon-autonomous transposon) and a plasmid comprising Tol2 transposase.

The term “non-autonomous transposon” in the present specification meansa transposon which is lost a transposase existed inside the transposonand cannot therefore perform its autonomous transfer. The non-autonomoustransposon can transfer the DNA inserted between transposon sequences ofthe non-autonomous transposon into the host cell chromosome, by allowinga transposase protein, an mRNA encoding the transposase protein or a DNAencoding the transposase protein to simultaneously present in the cell.

The transposase gene means a gene encoding a transposase. In order toimprove its expression efficiency in a mammalian cell, a sequence whichadjusts a space between the Kozak's consensus sequence (Kozak M.,Nucleic Acids Res., 12, 857-872 (1984)) or a ribosome binding sequence,Shine-Dalgarno sequence and the initiation codon, to an appropriatedistance (e.g., from 6 to 18 bases) may be connected to an upstream siteof the translation initiation codon ATG of the gene.

According to the method of the invention, in order to integrate a genefragment comprising a DNA encoding the protein of interest and aselectable marker gene in an expression vector into the chromosome of ahost cell, an expression vector which comprises the gene fragmentcomprising a DNA encoding the protein of interest and a selectablemarker gene and transposon sequences at both terminals of the genefragment is introduced into the host cell, and a transposase is allowedto act upon the transposon sequences comprised in the expression vectorwhich is introduced into the cell.

In order to allow a transposase to act upon the transposon sequencescomprised in the expression vector which is introduced into the cell,the transposase may be injected into the cell, or an expression vectorcomprising a DNA encoding the transposase may be introduced into thehost cell together with an expression vector comprising a DNA encodingthe protein of interest and a selectable marker gene. In addition, byintroducing an RNA encoding a transposase gene into the host cell, thetransposase may be expressed in the cell.

The expression vector is not particularly limited. Any expression vectorcan be used by optionally selecting from the expression vectors known tothose skilled in the art, depending on a host cell into which anexpression vector comprising a transposase gene is introduced; the use;and the like.

In order that a protein constituted from two or more polypeptides isproduced by the method of the invention, the DNA can be integrated intothe chromosome of the cell by integrating a DNA encoding the two or morepolypeptides into the same or different expression vectors and thenintroducing the expression vectors into a host cell.

The transposase may be inserted into an expression vector to expresstogether with the protein of interest or may be inserted into a vectordifferent from the expression vector. The transposase may be allowed toact transiently or may be allowed to act continuously, but it ispreferably to allow the transposase to act transiently in order toprepare a cell for stable production.

As the method for allowing the transposase to act transiently, examplesinclude a method comprising preparing an expression vector whichcomprises a DNA encoding the transposase and an expression vectorcomprising a DNA encoding a protein of interest and then introducingboth of the expression plasmids simultaneously into a host cell.

The term “expression vector” in the present specification means anexpression vector to be used for introducing a mammalian cell in orderto express a protein of interest. The expression vector used in theinvention has a structure in which at least a pair of transposonsequences is present at both sides of an expression cassette.

The term “expression cassette” in the present specification means anucleotide sequence which has a gene expression controlling regionnecessary for expressing a protein of interest and a sequence encodingthe protein of interest. Examples of the gene expression controllingregion include an enhancer, a promoter, and a terminator. The expressioncassette may contain a selectable marker gene.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, ametallothionein promoter, a heat shock promoter, SRa promoter, moloneymurine leukemia virus, an enhancer and the like. Also, the enhancer ofthe IE gene of human CMV can be used together with the promoter.

The “selectable marker gene” means an arbital other marker gene whichcan be used for distinguishing a cell to which a plasmid vector isintroduced from a cell lacking of the vector.

Examples of the selectable marker gene include a drug resistance gene (aneomycin resistance gene, a DHFR gene, a puromycin resistance gene, ablasticidin resistance gene, a hygromycin resistance gene, and acycloheximide resistance gene (Japanese Published Unexamined PatentApplication No. 262879/2002)), fluorescence and bio-luminescence markergenes (such as green fluorescent protein GFP) and the like.

In the invention, preferable selectable marker is a drug resistance geneand particularly preferable selectable marker is a cycloheximideresistance gene. In addition, by carrying out a gene modification of theselectable marker gene, drug resistance performance and luminescenceperformance of the selectable marker protein can also be modified.

Cycloheximide (hereinafter sometimes referred to as CHX) is a proteinsynthesis inhibitor, and as examples of the use of the CHX resistancegene as a selectable marker gene, the cases of yeast (Kondo K. J.Bacteriol., 177, 24, 7171-7177 (1995)) and animal cells (JapanesePublished Unexamined Patent Application No. 262879/2002) are known.

In the case of the animal cells, it has been found that the resistanceto cycloheximide is provided by a transformant which expresses a proteinencoded by the nucleotide sequence shown in SEQ ID NO:7 of SequenceListing in which proline at position 54 in human ribosomal proteinsubunit L36a encoded by the nucleotide sequence shown in SEQ ID NO:5 ofSequence Listing is substituted with glutamine.

The method for introducing the above-mentioned protein expression vectorcomprising a transposon sequence, a transposase expressing plasmidvector and RNA is not particularly limited. Examples include calciumphosphate transfection, electroporation, a liposome method, a gene gunmethod, lipofection and the like.

Examples of the method for directly introducing a transposase in theform of a protein include by microinjection or endocytosis for supplyinginto a cell. The gene transfer can be carried out by the methoddescribed in Shin Idenshi Kogaku Handbook (New Genetic EngineeringHandbook), edited by Masami Muramatsu and Tadashi Yamamoto, published byYodo-sha, ISBN 9784897063737.

The host cell may be any mammalian cell as long as it can be subculturedand stably express a protein of interest. Examples of the host cellinclude PER.C6 cell, human leukemia cell Namalwa cell, monkey cell COScell, rat myeloma cell YB2/3HL.P2.G11.16Ag.20 (also referred to asYB2/0), mouse myeloma cell NS0, mouse myeloma cell SP2/0-Ag14, Syrianhamster cell BHK, HBT5637 (Japanese Unexamined Patent ApplicationPublication No. 1998-000299), Chinese hamster ovarian cell CHO cell(Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad.Sci. USA., 601275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129(1973); Methods in Cell Science, 18, 115 (1996); Radiation Research,148, 260 (1997); Proc. Natl. Acad. Sci. USA., 77, 4216 (1980); Proc.Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular CellGenetics, Appendix I,II (pp. 883-900)), CHO/DG44, CHO-K1 (ATCC CCL-61),DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (LifeTechnologies, Cat #11619), Pro-3 and substrain of CHO cell.

In addition, the above-mentioned host cell can also be used in theprotein production method of the invention by modifying it so as to besuitable for the protein production, by modification of chromosomal DNA,introduction of an exogeneous gene, and the like.

Further, in order to control the sugar chain structure bound to aprotein of interest to be produced, Lec13 which acquired lectinresistance [Somatic Cell and Molecular Genetics, 12, 55 (1986)] and CHOcell from which α1,6-fucosyltransferase gene is deleted (WO2005/35586,WO2002/31140) can also be used as the host cell.

The protein of interest may be any protein so long as it can beexpressed by the method of the invention. Specifically, examples includea human serum protein, a peptide hormone, a growth factor, a cytokine, ablood coagulation factor, a fibrinolysis system protein, an antibody andpartial fragments of various proteins, and the like.

Preferable examples of the protein of interest include a monoclonalantibody such as a chimeric antibody, a humanized antibody and a humanantibody; Fc fusion protein; and albumin-bound protein; and a fragmentthereof.

An effector activity of a monoclonal antibody obtained by the method ofthe present invention can be controlled by various methods. For example,known methods are a method for controlling an amount of fucose(hereinafter, referred to also as “core fucose”) which is boundN-acetylglucosamine (GlcNAc) through α-1,6 bond in a reducing end of acomplex type N-linked sugar chain which is bound to asparagine (Asn) atposition 297 of an Fc region of an antibody (WO2005/035586,WO2002/31140, and WO00/61739), a method for controlling an effectoractivity of a monoclonal antibody by modifying amino acid group(s) of anFc region of the antibody, and the like. The effector activity of themonoclonal antibody produced by the method of the present invention canbe controlled by using any of the methods.

The “effector activity” means an antibody-dependent activity which isinduced via an Fc region of an antibody. As the effector activity, anantibody-dependent cellular cytotoxicity (ADCC activity), acomplement-dependent cytotoxicity (CDC activity), an antibody-dependentphagocytosis (ADP activity) by phagocytic cells such as macrophages ordendritic cells, and the like are known.

In addition, by controlling a content of core fucose of a complex typeN-linked sugar chain of Fc region of a monoclonal antibody, an effectoractivity of the antibody can be increased or decreased.

As a method for lowering a content of fucose which is bound to a complextype N-linked sugar chain bound to Fc of the antibody, an antibody towhich fucose is not bound can be obtained by the expression of anantibody using a CHO cell which is deficient in a gene encodingα1,6-fucosyltransferase. The antibody to which fucose is not bound has ahigh ADCC activity.

On the other hand, as a method for increasing a content of fucose whichis bound to a complex type N-linked sugar chain bound to Fc of anantibody, an antibody to which fucose is bound can be obtained by theexpression of an antibody using a host cell into which a gene encodingα1,6-fucosyltransferase is introduced. The antibody to which fucose isbound has a lower ADCC activity than the antibody to which fucose is notbound.

Further, by modifying amino acid residue(s) in an Fc region of anantibody, the ADCC activity or CDC activity can be increased ordecreased. For example, the CDC activity of an antibody can be increasedby using the amino acid sequence of the Fc region described inUS2007/0148165.

Further, the ADCC activity or CDC activity of an antibody can beincreased or decreased by modifying the amino acid as described in U.S.Pat. No. 6,737,056, or 7,297,775 or 7,317,091.

The term “suspension mammalian cell” in the present invention means acell which does not adhere to a cell culture anchorage coated forfacilitating adhesion of culture cells, such as microbeads, a culturecontainer for tissue culture (also referred to as a tissue culture oradhesion culture container and the like) and the like, and can surviveand grow by suspending in the culture liquid.

When the cell does not adhere to the cell culture anchorage, it maysurvive and grow under a state of a single cell in the culture liquid orsurvive and grow under a state of a cell mass formed by theagglutination of two or more cells.

In addition, as the suspension mammalian cell to be used in the presentinvention, a cell which can survive and grow in a serum-free medium thatdoes not contain fetal calf serum (hereinafter referred to as FCS) andthe like, while suspending in the culture liquid without adhering to thecell culture anchorage, is preferable, and a mammalian cell which cansurvive and grow while suspending in a protein-free medium that does notcontain protein is more preferable.

As the culture container for tissue culture, it may be any culturecontainer such as a flask, a Petri dish and the like, so long as coatingfor adhesion culture is applied thereto. Specifically, for example,whether or not it is a suspension mammalian cell can be confirmed by theuse of commercially available tissue culture flask (manufactured byGreiner), adhesion culture flask (manufactured by Sumitomo Bakelite) andthe like.

As the suspension mammalian cell to be used in the present invention, itmay be either a cell prepared by further adapting a cell originallyhaving a suspension property to suspension culture or a suspensionmammalian cell prepared by adapting an adhesive mammalian cell tosuspension culture conditions.

Examples of the cell originally having a suspension property includePER.C6 cell, a rat myeloma cell YB2/3HL.P2.G11.16Ag.20 (or also calledYB2/0), CHO-S cell (manufactured by Invitrogen) and the like.

The aforementioned “suspension mammalian cell prepared by adapting anadhesive mammalian cell to suspension culture conditions” can beprepared by the method described in Mol. Biotechnol., 2000, 15(3),249-57 or by the method shown in the following, and can be prepared byestablishing a cell which shows proliferation property and survivingproperty similar to those before the suspension culture adaptation orsuperior to those before adapting to suspension culture (J. Biotechnol.,2007, 130(3), 282-90).

The term “similar to those before the suspension culture adaptation”means that survival ratio, proliferation rate (doubling time) and thelike of the cell adapted to the suspension culture are substantially thesame as those of the cell before adapting suspension culture.

Examples of the method for adapting an adhesive mammalian cell tosuspension culture conditions according to the present invention includethe following method. The serum content of a serum-containing medium isreduced to 1/10 and sub-culturing is repeated at relatively highconcentration of cell. When the mammalian cell comes to be able tosurvive and proliferate, the serum content is further reduced and thesub-culturing is repeated. By this method, a suspension mammalian cellwhich can survive and proliferate under serum-free conditions can beprepared.

In addition, a suspension mammalian cell can also be prepared by amethod comprising culturing with the addition of an appropriate nonionicsurfactant such as Pluronic-F68 or the like in the culture liquid.

Examples of the adhesive mammalian cell which acquires suspensionproperty by adapting to a suspension culture condition include a mousemyeloma cell NS0, a CHO cell and the like.

In the present invention, as a property possessed by the suspensionmammalian cell, when 2×10⁵ cells/ml of the cell is suspension-cultured,the cell concentration after culturing for 3 or 4 days is preferably5×10⁵ cells/ml or more, more preferably 8×10⁵ cells/ml or more,particularly preferably 1×10⁶ cells/ml or more, most preferably 1.5×10⁶cells/ml or more.

In addition, doubling time of the suspension mammalian cell of thepresent invention is preferably 48 hours or less, more preferably 24hours or less, particularly preferably 18 hours or less, most preferably11 hours or less.

Examples of the medium for suspension culturing include commerciallyavailable media, such as CD-CHO medium (manufactured by Invitrogen),EX-CELL 325-PF medium (manufactured by SAFC Biosciences), SFM4CHO medium(manufactured by HyClone) and the like. In addition, it can also beobtained by mixing saccharides, amino and the like acids which arenecessary for the culturing of mammalian cells.

The suspension mammalian cell can be cultured using a culture containerwhich can be used for suspension culturing under a culture conditioncapable of suspension culturing. Examples of the culture containerinclude a 96 well plate for cell culture (manufactured by Corning), aT-flask (manufactured by Becton Dickinson), an Erlenmeyer flask(manufactured by Corning) and the like.

Regarding the culture conditions, for example, it can be staticallycultured in an atmosphere of 5% CO₂ at a culture temperature of 37° C. Ashaking culture equipment, such as culturing equipment for suspensionculture exclusive use, Wave Bioreactor (manufactured by GE HealthcareBioscience), can also be used.

Regarding the suspension culture conditions of a suspension mammaliancell using the Wave Bioreactor equipment, the cell can be cultured bythe method described on the GE Healthcare Bioscience homepagewww.gelifesciences.co.jp/tech-support/manual/pdf/cellcult/wave-03-16.pdf.

In addition to the shaking culture, culturing by a rotation agitationequipment such as a bioreactor, can also be used. Culturing using abioreactor can be carried out by the method described in Cytotechnology,(2006) 52: 199-207, and the like.

In the present invention, when a cell line other than the suspensionmammalian cells is used, any cell line can be used so long as it is amammalian cell line adapted to the suspension culture by theabove-mentioned method and is a cell line which can be used in theprotein producing method of the present invention.

Purification of the protein of interest produced by the suspensionmammalian cell is carried out by separating the protein of interest fromimpurities other than the protein of interest in a culture liquid orcell homogenate containing the protein of interest. Examples of theseparation method include centrifugation, dialysis, ammonium sulfateprecipitation, column chromatography, a filter and the like. Theseparation can be carried out based on the difference in physicochemicalproperties of the protein of interest and impurities and based on thedifference in their affinity for the column carrier.

The method for purifying the protein of interest can be carried out, forexample, by the method described in Protein Experimentation Note (thefirst volume)—Extraction, Separation and Expression of RecombinantProtein (translation of a textbook written in Japanese) (edited byMasato Okada and Kaori Miyazaki, published by Yodo-sha, ISBN9784897069180).

The entire contents of the references, such as the scientific documents,patents, patent applications cited herein are incorporated herein byreference to the same degree of those illustratively described,respectively.

The present invention has been described in the foregoing by showingpreferred embodiments thereof for the sake of easy understanding.Hereinafter, the present invention is further described specificallybased on examples, but the above-mentioned explanations and thefollowing examples are provided merely for the purpose ofexemplifications and not provided for the purpose of limiting theinvention. Accordingly, the scope of the invention is not limited to theembodiments and examples which are specifically described herein, but islimited by the claims alone.

Various experimental techniques relating to genetic recombinationdescribed hereinafter, such as the cloning and the like were carried outin accordance with the genetic engineering techniques described inMolecular Cloning 2^(nd) edition edited by J. Sambrook, E. F. Frisch andT. Maniatis, Current Protocols in Molecular Biology edited by FrederickM. Ausubel et al, published by Current Protocols, and the like.

By the method for producing the protein of the present invention, aprotein of interest can be efficiently produced using a suspensionmammalian cell. The cell of the present invention can be used as aprotein producing cell for producing a recombinant protein.

EXAMPLES Example 1

Preparation of Transposon Vector for Expressing Anti-Human Influenza M2Antibody

A plasmid which contains a gene expression cassette for mammalian cellscomprising an arbitrary human antibody gene and a drug resistance markergene inserted between a pair of Tol2 transposon sequences was used as aplasmid vector for protein expression.

Each DNA of the used genes was chemically and artificially synthesizedbased on a known nucleotide sequence or obtained by preparing primersfor its both terminal sequences and then carrying out PCR using anappropriate DNA source as a template. In order to carry out the genemanipulation later, a restriction site for a restriction enzyme wasadded to the terminal of the primer.

Among the nucleotide sequence of the non-autonomous Tol2 transposonedisclosed by Japanese Published Unexamined Patent Application No.235575/2003 (SEQ ID NO:1), the nucleotide sequence at position 1 to 200(Tol2-L sequence) (SEQ ID NO:2) and the nucleotide sequence at positions2285 to 2788 (Tol2-R sequence) (SEQ ID NO:3) were used as the transposonsequences.

Each synthetic DNA fragments comprising a pair of transposon sequences(manufactured by TAKARA BIO INC.) was prepared by the following method.A DNA fragment comprising a nucleotide sequence in which a recognitionsequence of a restriction enzyme NruI was attached to both of the5′-terminal and 3′-terminal of the Tol2-R sequence was prepared. Then, aDNA fragment comprising a nucleotide sequence in which a recognitionsequence of a restriction enzyme FseI was attached to the 5′-terminal ofthe Tol2-L sequence and a restriction enzyme AscI was attached to the3′-terminal thereof was prepared.

Next, the thus prepared DNA fragments comprising Tol2-R sequence andTol2-L sequence were inserted into an expression vector N5LG1-M2-Z3vector (WO2006/061723) comprising a nucleotide sequence encoding anamino acid sequence of anti-human influenza M2 antibody Z3G1.

The N5LG1-M2-Z3 vector (WO2006/061723) into which a nucleotide sequence(SEQ ID NO:8) encoding the H chain of the anti-human influenza M2antibody Z3G1 (ATCC Deposit No. PTA-5968: deposited Mar. 13, 2004,American Type Culture Collection, Manassas, Va., USA) and a nucleotidesequence (SEQ ID NO:10 and SEQ ID NO:11) encoding the L chain (SEQ IDNO:9) of the same were inserted under the control of the CMVenhancer/promoter control was used as an antibody gene expressioncassette.

The DNA fragment comprising the Tol2-R sequence was inserted into therestriction enzyme NruI site of the N5LG1-M2-Z3 vector, at the5′-terminal side of a gene fragment comprising the antibody geneexpression cassette and a resistance marker gene. Then, the DNA fragmentcomprising the Tol2-L sequence was inserted into the restriction enzymeFseI and AscI sites at the 3′-terminal side.

In addition, a transposon vector for expressing an anti-human influenzaM2 antibody was constructed (FIG. 1) by inserting a cycloheximideresistance gene expression cassette connected with a nucleotide sequence(SEQ ID NO:5) encoding a resistance gene for cycloheximide (a gene inwhich proline at position 54 of the human ribosomal protein L36a wassubstituted with glutamine) into the FseI recognition site of theN5LG1-M2-Z3 vector connected with the Tol2 transposon sequence, underthe control of the CMV enhancer/promoter.

On the other hand, a vector containing no transposon sequences was namedanti-human influenza M2 antibody expression vector and used as thecontrol vector (FIG. 2).

Example 2

Preparation of Transposase Expression Vector

The transposase was expressed using an expression vector independent ofthe expression vector of the antibody of interest. That is, a gene whichis encoding a medaka fish-derived Tol2 transposase (SEQ ID NO:4) wasinserted into a downstream of the CAGGS promoter of a pCAGGS vector(Gene, 108, 193-200, 1991) and used as the expression vector (FIG. 3).

Example 3

(1) Preparation of Suspension CHO Cell

An adhesive CHO cell which had been cultured using an α-MEM medium(manufactured by Invitrogen) containing 10% serum (FCS) was peeled offand recovered by a trypsin treatment and shaking-cultured at 37° C. in a5% CO₂ incubator using fresh α-MEM medium containing 10% FCS. Severaldays thereafter, growth of these cells was confirmed and then shakingculture was carried out by seeding them into a α-MEM medium containing5% FCS at a concentration of 2×10⁵ cells/ml.

Further several days thereafter, the inoculation was similarly carriedout using the α-MEM medium containing 5% FCS. Finally, a cell adapted tothe suspension culture was prepared by repeating the sub-culture andshaking culture using serum-free α-MEM medium and confirming that thecells have the same growing ability of the case of their culturing inthe presence of serum.

(2) Preparation of Antibody-Producing CHO Cell

The transposon vector for expressing the anti-human influenza M2antibody prepared in Example 1 and Example 2 (hereinafter referred to astransposon vector) and Tol2 transposase expression vector pCAGGS-T2TP(FIG. 3, Kawakami K. & Noda T., Genetics, 166, 895-899 (2004)) were usedas the expression vectors. In addition, the anti-human influenza M2antibody expression vector having no transposon sequences was used asthe control.

By introducing the aforementioned expression vectors into the suspensionculture-adapted CHO-K1 cell (American Type Culture Collection Cat. No.CCL-61) or HEK293 cell (FreeStyle 293F cell, manufactured byInvitrogen), the frequencies of obtaining cycloheximide-resistant cloneswere compared.

Each cells (4×10⁶ cells) was suspended in 400 μl of PBS, and thetransposon vector for expressing the anti-human influenza M2 antibody(10 μg) and Tol2 transposase expression vector (25 μg) wereco-transfected directly in the form of circular DNA by electroporation.In this connection, in order to express the Tol2 transposasetransiently, the Tol2 transposase expression vector was directlyintroduced in the form of circular DNA for the purpose of preventingfrom integrating into the host chromosome.

In addition, as the control, the anti-human influenza M2 antibodyexpression vector (10 μg) was linearized by a restriction enzyme andthen introduced into each cells, in accordance with the standard genetransfer method by electroporation.

The electroporation was carried out using a cuvette of 4 mm in gap width(manufactured by Bio-Rad®), using an electroporator (Gene Pulser Xcell™System (manufactured by Bio-Rae)) under conditions of 300 V in voltage,500 μF in electrostatic capacity and room temperature.

After the transfection by electroporation, each cell was seeded intothree 96-well plates and cultured in a CO₂ incubator for 3 days usingthe EX-CELL 325-PF medium manufactured by SAFC Biosciences for the CHOcell, and the FreeStyle-293 medium (manufactured by Invitrogen) for theHEK293 cell.

Next, from the day of medium exchange on the 4th day of thetransfection, 3 μg/ml of cycloheximide was added to the medium so thatthe cells were cultured in the presence of cycloheximide, followed byculturing for 3 weeks while carrying out the medium exchange in everyweek.

After culturing for 3 weeks, the number of wells in whichcycloheximide-resistant colonies were found was counted. The results areshown in Table 1 and Table 2.

TABLE 1 Comparison of the numbers of cycloheximide- resistant cells (CHOcell) Transposon vector Conventional vector Test 1 155/288 0/288 Test 2100/288 0/288 Test 3  94/288 0/288

TABLE 2 Comparison of the numbers of cycloheximide- resistant cells(HEK293 cell) Transposon vector Conventional vector Test 1 0/288 0/288Test 2 0/288 0/288 Test 3 0/288 0/288

As shown in Table 1, each the anti-human influenza M2 antibodyexpression transposon vector or anti-human influenza M2 antibodyexpression vector was introduced into the suspension CHO-K1 cell. As aresult, cycloheximide-resistant transformants were not obtained from thecell introduced with anti-human influenza M2 antibody expression vectorlike the case of other cell lines, but cycloheximide-resistanttransformants were obtained from the cell introduced with transposonvector for expressing anti-human influenza M2 antibody with a highfrequency.

On the other hand, as shown in Table 2, cycloheximide-resistanttransformants were not obtained when either of the transposon vector forexpressing anti-human influenza M2 antibody and anti-human influenza M2antibody expression vector was introduced into the HEK293 cell.

Based on these results, it was found that the intended protein-encodedgene and cycloheximide resistance gene which were inserted between apair of transposon sequences are efficiently introduced into thechromosome of the host cell, namely a suspension mammalian cell.

(3) Examination on the Antibody Production by Suspension CHO Cell andAdhesive CHO Cell

In order to examine antibody production efficiency by a suspension CHOcell or an adhesive CHO cell, the amounts of antibodies produced byrespective cell lines were examined. As the suspension CHO cell, thesuspension CHO-K1 cell adapted to suspension culture was used. Inaddition, as the adhesive CHO cell, the adhesive CHO-K1 cell beforeadaptation to suspension culture was used.

The anti-human influenza M2 antibody expression transposon vector (10μg) and Tol2 transposase expression vector (25 μg) were introduced intothe suspension CHO-K1 cell and adhesive CHO-K1 cell by means ofelectroporation, respectively. Thereafter, the suspension CHO-K1 celland the adhesive CHO-K1 cell were seeded into three 96-well plates foreach cell.

A medium for suspension cells (EX-CELL 325-PF, manufactured by SAFCBiosciences) was used for the suspension CHO-K1 cell, and the α-MEMmedium containing 10% serum was used for the adhesive CHO-K1 cell. Eachcell was cultured in a CO₂ incubator for 3 days. From the day of mediumexchange on the 4th day of the transfection, 3 μg/ml of cycloheximidewas added to the medium so that the cells were cultured in the presenceof cycloheximide and the cells were further cultured for 3 weeks. Inthis case, the medium exchange was carried out every week.

For the suspension CHO-K1 cell, 1×10⁶ of the cells were seeded into a6-well plate and shaking-cultured in a CO₂ incubator for 3 days, and theamount of the anti-human influenza M2 antibody protein was measured byHPLC using the culture supernatant.

For the adhesive CHO-K1 cell, medium exchange was carried out when thecell reached confluent on a 6-well plate (2×10⁶ cells), and 3 days afterstatic culture, the amount of the antibody protein was measured by HPLCusing the culture supernatant.

The antibody concentration in the culture supernatant was measured inaccordance with the method described in Yeast Res., 7 (2007), 1307-1316.The results are shown in FIG. 4A and FIG. 4B.

As shown in FIG. 4A, a large number of cells showing a markedly highantibody expression level were obtained when the CHO-K1 cell adapted tosuspension culture was used. On the other hand, as shown in FIG. 4B,only the cells showing an expression level of the HPLC detection limit(5 μg/ml) or less were obtained when the adhesive CHO-K1 cell was used.

Based on these results, it was found that, for the expression of aprotein of interest using a transposon vector, the protein of interestcan be expressed at a high level when a suspension mammalian cell isused.

In addition, it was found from the results of Examples 1 to 3 that themethod of the invention can be used as a novel method for producing aprotein of interest, by efficiently preparing a production cell whichcan highly express an exogeneous gene using a suspension mammalian celladapted to suspension culture.

Example 4

Preparation of Tol1 Transposon Vector for Expressing Anti-HumanInfluenza M2 Antibody

In the same manner as in Example 1, a plasmid which contains a geneexpression cassette for mammalian cells, comprising an arbitrary humanantibody gene and a drug resistance marker gene inserted between a pairof Tol1 transposon sequences, was used as a protein expression plasmidvector.

Each DNA of the used genes was chemically synthesized artificially basedon the known sequence information or obtained by preparing primers ofits both terminal sequences and carrying out PCR using an appropriateDNA source as the template. For the gene manipulation to be carried outlater, a site cleaved by a restriction enzyme was added to the end ofthe primer.

Among the non-autonomous Tol1 transposon nucleotide sequence shown inSEQ ID NO:13 of Sequence Listing (WO2008/072540), the nucleotidesequence at positions 1 to 200 (Tol1-L sequence) (SEQ ID NO:14) and thenucleotide sequence at positions 1351 to 1855 (Tol1-R sequence) (SEQ IDNO:15) were used as the transposon sequences.

Each of the synthetic DNA fragments comprising each a pair of transposonsequences was prepared by the following method. A DNA fragmentcomprising a nucleotide sequence in which a recognition sequence of arestriction enzyme NruI was connected to both of the 5′-terminal and3′-terminal of the Tol1-R sequence. Then, a DNA fragment comprising anucleotide sequence in which a recognition sequence of a restrictionenzyme FseI was connected to the 5′-terminal of the Tol1-L sequence anda restriction enzyme AscI was connected to the 3′-terminal thereof.

Next, the thus prepared DNA fragments comprising Tol1-R sequence andTol1-L sequence were inserted into the expression vector N5LG1-M2-Z3vector. The DNA fragment comprising the Tol1-R sequence was insertedinto the restriction enzyme NruI site of the N5LG1-M2-Z3 vector,existing on the 5′-terminal side of a gene fragment comprising theantibody gene expression cassette and a resistance marker gene, and theDNA fragment comprising the Tol1-L sequence was inserted into therestriction enzyme FseI and AscI sites existing on the 3′-terminal side.

In addition, Tol1 transposon vector for expressing an anti-humaninfluenza M2 antibody was constructed (FIG. 5) by inserting acycloheximide resistance gene expression cassette connected with aresistance gene for cycloheximide (a gene in which proline at position54 in the human ribosomal protein L36a was mutated to glutamine) intothe FseI recognition site of the N5LG1-M2-Z3 vector connected with theTol1 transposon sequence, under the control of the CMVenhancer/promoter.

Example 5

Preparation of Tol1 Transposase Expression Vector

The transposase was expressed using an expression vector independentfrom the expression vector of the antibody of interest. That is, a Tol1transposase gene expression cassette connected with a DNA fragmentencoding a medaka fish-derived Tol1 transposase, containing thenucleotide sequence shown in SEQ ID NO:16 of Sequence Listing, wasinserted into pBluescriptII SK (+) (manufactured by Stratagene) underthe CMV enhancer/promoter control and used as the expression vectorpTol1ase (FIG. 6).

Example 6

(1) Preparation of Antibody-Producing CHO Cell

The Tol1 transposon vector for expressing the anti-human influenza M2antibody (hereinafter referred to as Tol1 transposon vector) and Tol1transposase expression vector pTol1ase of Example 4 and Example 5 wereused as the expression vectors. In addition, the CHO-K1 cell prepared byadapting to suspension culture in the same manner as in Example 3(1) wasused as the cell.

The aforementioned expression vectors were introduced into the CHO-K1cell adapted to suspension culture, and the frequency of obtainingclones resistant to cycloheximide was measured. The CHO-K1 cell adaptedto suspension culture (4×10⁶ cells) were suspended in 400 μl of PBS, andthe Tol1 transposon vector for expressing the anti-human influenza M2antibody (10 μg) and Tol1 transposase expression vector (50 μg) wereco-transfected directly in the form of circular DNA by electroporation.In order to effect transient expression of the Tol1 transposase, theTol1 transposase expression vector was directly introduced in the formof circular DNA for the purpose of preventing from integrating into thehost chromosome.

The electroporation was carried out using a cuvette of 4 mm in gap width(manufactured by Bio-Rad®), using an electroporator (Gene Pulser Xcell™System (manufactured by Bio-Rae)) under conditions of 300 V in voltage,500 μF in electrostatic capacity and room temperature.

After the transfection by electroporation, each cell was seeded into two96-well plates and cultured in a CO₂ incubator for 3 days using theEX-CELL 325-PF medium (manufactured by SAFC Biosciences) for the CHOcell. Next, from the day of medium exchange on the 4th day of thetransfection, 3 μg/ml of cycloheximide was added to the medium so thatthe cells were cultured in the presence of cycloheximide, followed byculturing for 3 weeks while carrying out the medium exchange every week.

After the culturing for 3 weeks, the number of wells in whichcycloheximide-resistant colonies were found was counted. The results areshown in Table 3. Each of the tests 1 to 3 in Table 3 shows a result ofcarrying out the gene transfer three times.

TABLE 3 Tol1 transposon vector Tests 1 133/192 Tests 2  67/192 Tests 3122/192

As shown in Table 3, when the Tol1 transposon vector for expressing theanti-human influenza M2 antibody was introduced into the suspensionCHO-K1 cell, cycloheximide-resistant transformants were obtained at ahigh frequency similarly to Example 3 in which the Tol2 transposonvector for expressing the anti-human influenza M2 antibody wasintroduced.

It was found based on these results that the antibody gene andcycloheximide resistance gene inserted between a pair of transposonsequences are efficiently transduced into the chromosome of the hostcell, namely the suspension mammalian cell, in the case of using theTol1 transposon, too.

(2) Examination on Antibody Production by Suspension CHO-K1 Cell

Antibody production efficiency of the suspension CHO-K1 cell wasexamined using the suspension CHO-K1 cell. The transposon vector forexpressing the anti-human influenza M2 antibody (10 μg) and Tol1transposase expression vector (50 μg) were introduced by electroporationinto the suspension CHO-K1 cell adapted to suspension culture.

Thereafter, the cells were seeded into respective two 96-well plates andcultured for 3 days in a CO₂ incubator using the suspension culturemedium EX-CELL 325-PF. From the medium exchange on the 4th days afterthe electroporation, the cells were cultured for 3 weeks in the presenceof 3 μg/ml of cycloheximide. In this case, the medium exchange wascarried out every week.

For the suspension CHO-K1 cell, 1×10⁶ of the cells were seeded into a6-well plate and shaking-cultured in a CO₂ incubator for 3 days, andamount of the anti-human influenza M2 antibody protein was measured byHPLC using the culture supernatant.

The antibody concentration in culture supernatant was measured inaccordance with the method described in Yeast Res., 7 (2007), 1307-1316.The results are shown in FIG. 7.

As shown in FIG. 7, a large number of cells showing a markedly highantibody expression level were obtained in the case of the use of theTol1 transposon, too. From this result, it was found that similar to thecase of the use of the Tol2 transposon-derived nucleotide sequence, asuspension mammalian cell capable of highly expressing the protein ofinterest can also be obtained when a Tol1 transposon-derived nucleotidesequence is used as the transposon sequence.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese application No. 2009-140626, filedon Jun. 11, 2009, and U.S. provisional application No. 61/186,138, filedon Jun. 11, 2009, the entire contents of which are incorporated hereintoby reference. All references cited herein are incorporated in theirentirety.

What is claimed is:
 1. A method selected from the group consisting of(I) and (II): (I) a method for increasing expression of a protein ofinterest, comprising introducing an expression vector which comprises agene fragment comprising a DNA encoding the protein and a selectablemarker gene, and transposon sequences at both terminals of the genefragment, into a suspension mammalian cell; integrating the genefragment inserted between a pair of the transposon sequences, into achromosome of the mammalian cell to obtain a mammalian cell capable ofexpressing the protein; and suspension-culturing the mammalian cell; or(II) a method for increasing expression of a protein of interest, whichcomprises the following steps (A) to (C): (A) a step of simultaneouslyintroducing the following expression vectors (a) and (b) into asuspension mammalian cell, (a) an expression vector which comprises agene fragment comprising a DNA encoding the protein, and transposonsequences at both terminals of the gene fragment, (b) an expressionvector which comprises a DNA encoding a transposase which recognizes thetransposon sequences and has activity of transferring a gene fragmentinserted between a pair of the transposon sequences into a chromosome,(B) a step of transiently expressing the transposase from the expressionvector introduced in the step (A) to integrate the gene fragmentinserted between a pair of the transposon sequences into a chromosome ofthe mammalian cell, to obtain a suspension mammalian cell capable ofexpressing the protein, and (C) a step of suspension-culturing thesuspension mammalian cell capable of expressing the protein obtained instep (B); wherein in the methods of (I) or (II), the pair of transposonsequences are nucleotide sequences derived from a pair of Tol1transposons or nucleotide sequences derived from a pair of Tol2transposons, and wherein the suspension mammalian cell is at least oneselected from a suspension CHO cell in which a CHO cell is adapted tosuspension culture, a PER.C6 cell, a rat myeloma cellYB2/3HL.P2.G11.16Ag.20 (or YB2/0) and a suspension mouse myeloma cellNS0 adapted to suspension culture.
 2. The method according to claim 1,(I) wherein the suspension mammalian cell is a cell capable of survivingand proliferating in a serum-free medium; (II) wherein the selectablemarker gene is a cycloheximide resistance gene; and/or (III) wherein thepair of transposon sequences are nucleotide sequences derived from apair of DNA-type transposons which function in a mammalian cell.
 3. Themethod according to claim 1, wherein the CHO cell is at least oneselected from CHO-K1, CHO-K1SV, DUKXB11, CHO/DG44, Pro-3 and CHO-S. 4.The method according to claim 2, wherein the cycloheximide resistancegene is a gene encoding a mutant of human ribosomal protein L36a.
 5. Themethod according to claim 4, wherein the mutant is a mutant in whichproline at position 54 of the human ribosomal protein L36a issubstituted with other amino acid.
 6. The method according to claim 5,wherein the other amino acid is glutamine.
 7. The method according toclaim 1, (I) wherein the nucleotide sequences derived from a pair ofTol2 transposons are a nucleotide sequence comprising the nucleotidesequence shown in SEQ ID NO:2 and the nucleotide sequence shown in SEQID NO:3; or (II) wherein the nucleotide sequences derived from a pair ofTol1 transposons are the nucleotide sequence shown in SEQ ID NO:14 andthe nucleotide sequence shown in SEQ ID NO:15.
 8. A suspension mammaliancell capable of increasing expression of a protein of interest, (I) intowhich an expression vector comprising a gene fragment comprising a DNAencoding the protein and a selectable marker gene, and transposonsequences at both terminals of the gene fragment is introduced, tointegrate the gene fragment inserted between a pair of the transposonsequences into a chromosome; or (II) into which an expression vector (a)comprising a gene fragment comprising a DNA encoding the protein and aselectable marker gene, and transposon sequences at both terminals ofthe gene fragment, and an expression vector (b) comprising a DNAencoding a transposase which recognizes the transposon sequences and hasactivity of transferring the gene fragment inserted between a pair ofthe transposon sequences into a chromosome to integrate the genefragment inserted between a pair of the transposon sequences into thechromosome, wherein in the methods of (I) or (II), the pair oftransposon sequences are nucleotide sequences derived from a pair ofTol1 transposons or nucleotide sequences derived from a pair of Tol2transposons, and wherein the suspension mammalian cell is at least oneselected from a suspension CHO cell in which a CHO cell is adapted tosuspension culture, a PER.C6 cell, a rat myeloma cellYB2/3HL.P2.G11.16Ag.20 (or YB2/0) and a suspension mouse myeloma cellNS0 adapted to suspension culture.
 9. The cell according to claim 8, (I)wherein the cell is a cell capable of surviving and proliferating in aserum-free medium; (II) wherein the selectable marker gene is acycloheximide resistance gene; and/or (III) wherein the pair oftransposon sequences are nucleotide sequences derived from a pair of DNAtype transposons which function in a mammalian cell.
 10. The cellaccording to claim 9, wherein the CHO cell is at least one selected fromCHO-K1, CHO-K1SV, DUKXB11, CHO/DG44, Pro-3 and CHO-S.
 11. The cellaccording to claim 9, wherein the cycloheximide resistance gene is agene encoding a mutant of human ribosomal protein L36a.
 12. The cellaccording to claim 11, wherein the mutant is a mutant in which prolineat position 54 of the human ribosomal protein L36a is substituted withother amino acid.
 13. The cell according to claim 12, wherein the otheramino acid is glutamine.
 14. The cell according to claim 8, (I) whereinthe nucleotide sequences derived from a pair of Tol2 transposons are thenucleotide sequence shown in SEQ ID NO:2 and the nucleotide sequenceshown in SEQ ID NO:3; or (II) wherein the nucleotide sequences derivedfrom a pair of Tol1 transposons are the nucleotide sequence shown in SEQID NO:14 and the nucleotide sequence shown in SEQ ID NO:15.
 15. Theexpression vector for increasing expression of a protein of interestaccording to claim 1 or 8, comprising a gene fragment comprising a DNAencoding a protein of interest and a selectable marker gene, and a pairof transposon sequences at both terminals of the gene fragment; andwherein the pair of transposon sequences are nucleotide sequencesderived from a pair of Tol1 transposons or nucleotide sequences derivedfrom a pair of Tol2 transposons.
 16. The expression vector according toclaim 15, (I) wherein the nucleotide sequences derived from a pair ofthe Tol2 transposons are the nucleotide sequence shown in SEQ ID NO:2and the nucleotide sequence shown in SEQ ID NO:3; or (II) wherein thenucleotide sequences derived from a pair of the Tol1 transposons are thenucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequenceshown in SEQ ID NO:15.